Polycyclic aromatic hydrocarbons (PAHs) are widespread in various ecosystems and are pollutants of great concern due to their potential toxicity, mutagenicity and carcinogenicity. Because of their hydrophobic nature, most PAHs bind to particulates in soil and sediments, rendering them less available for biological uptake. Microbial degradation represents the major mechanism responsible for the ecological recovery of PAH-contaminated sites. The goal of this review is to provide an outline of the current knowledge of microbial PAH catabolism. In the past decade, the genetic regulation of the pathway involved in naphthalene degradation by different gram-negative and gram-positive bacteria was studied in great detail. Based on both genomic and proteomic data, a deeper understanding of some high-molecular-weight PAH degradation pathways in bacteria was provided. The ability of nonligninolytic and ligninolytic fungi to transform or metabolize PAH pollutants has received considerable attention, and the biochemical principles underlying the degradation of PAHs were examined. In addition, this review summarizes the information known about the biochemical processes that determine the fate of the individual components of PAH mixtures in polluted ecosystems. A deeper understanding of the microorganism-mediated mechanisms of catalysis of PAHs will facilitate the development of new methods to enhance the bioremediation of PAH-contaminated sites.

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Chemical synthesis of DNA sequences provides a powerful tool for modifying genes and for studying gene function, structure and expression. Here, we report a simple, high-fidelity and cost-effective PCR-based two-step DNA synthesis (PTDS) method for synthesis of long segments of DNA. The method involves two steps. (i) Synthesis of individual fragments of the DNA of interest: ten to twelve 60mer oligonucleotides with 20 bp overlap are mixed and a PCR reaction is carried out with high-fidelity DNA polymerase Pfu to produce DNA fragments that are approximately 500 bp in length. (ii) Synthesis of the entire sequence of the DNA of interest: five to ten PCR products from the first step are combined and used as the template for a second PCR reaction using high-fidelity DNA polymerase pyrobest, with the two outermost oligonucleotides as primers. Compared with the previously published methods, the PTDS method is rapid (5-7 days) and suitable for synthesizing long segments of DNA (5-6 kb) with high G + C contents, repetitive sequences or complex secondary structures. Thus, the PTDS method provides an alternative tool for synthesizing and assembling long genes with complex structures. Using the newly developed PTDS method, we have successfully obtained several genes of interest with sizes ranging from 1.0 to 5.4 kb.

Branched-chain fatty acids (BCFAs) typically constitute more than 90 % of the fatty acids of Listeria monocytogenes. The authors have previously described two Tn917-induced, cold-sensitive, BCFA-deficient (<40 %) L. monocytogenes mutants (cld-1 and cld-2) with lowered membrane fluidity. Sequence analyses revealed that Tn917 was inserted into different genes of the branched-chain a-keto acid dehydrogenase cluster (bkd) in these two mutants. The cold-sensitivity and BCFA deficiency of cld-1, in which Tn917 was inserted into bkdB, were complemented in trans by cloned bkdB. The growth and corresponding BCFA content of the mutants at 37 6C were stimulated by fatty acid precursors bypassing Bkd, 2-methylbutyrate (precursor for odd-numbered anteiso-fatty acids), isobutyrate (precursor for even-numbered iso-fatty acids) and isovalerate (precursor for odd-numbered iso-fatty acids). In contrast, the corresponding Bkd substrates, a-ketomethylvalerate, a-ketoisovalerate and a-ketoisocaproate, exhibited much poorer activity. At 26 6C, 2-methylbutyrate and isovalerate stimulated the growth of the mutants, and at 10 6C, only 2-methylbutyrate stimulated growth. Pyruvate depressed the BCFA content of cld-2 from 33 % to 27 %, which may be close to the minimum BCFA requirement for L. monocytogenes. The transcription of bkd was enhanced by Bkd substrates, but not by low temperature. When provided with the BCFA precursors, cld-2 was able to increase its anteiso-C 15 : 0 fatty acid content at 10 6C compared to 37 6C, which is the characteristic response of L. monocytogenes to low temperature. This implies that Bkd is not the major cold-regulation point of BCFA synthesis. INTRODUCTIONListeria monocytogenes causes the potentially life-threatening infection known as listeriosis, which has a fatality rate of 20-25 % (Mead et al., 1999). Listeriosis outbreaks have been linked to the consumption of food contaminated with this organism. Nowadays, there is an increased consumption of ready-to-eat, chilled and frozen foods, with decreased use of chemical microbial control agents. The ability of L. monocytogenes to grow at refrigeration temperatures is a critical aspect of its role as a food-borne pathogen, since refrigeration temperature inhibits the growth of most other food-borne pathogens (Bryan, 2004). However, the underlying psychrotolerance mechanism is not completely understood, and a better understanding may result in a novel strategy for controlling the growth of L. monocytogenes at low temperatures.Low temperature has profound effects on all aspects of microbial cell structure and function, involving the structural integrity of macromolecules, macromolecular assemblies, protein synthesis and nutrient uptake (Panoff et al., 1998;Weber & Marahiel, 2002 Two Tn917 transposon-induced mutants (cld-1 and cld-2) have lost the ability to grow at 4 u C on solid media, but are not defective in the induction of cold-shock proteins (Bayles et al., 1996). These mutants are deficient in the production of odd-numbered BCFAs, and exhibit atypical am...